Method for isolation of soluble polypeptides

ABSTRACT

Polypeptides with desirable biophysical properties such as solubility, stability, high expression, monomericity, binding specificity or non-aggregation, including monomeric human VHs and VLs, are identified using a high throughput method for screening polypeptides, comprising the steps of obtaining a phage display library, allowing infection of a bacterial lawn by the library phage, and identifying phage which form larger than average plaques on the bacterial lawn. Sequences of monomeric human VHs and VLs are identified, which may be useful for immunotherapy or as diagnostic agents. Multimer complexes of human VHs and VLs are also identified. The VHs and VLs identified may be used to create further libraries for identifying additional polypeptides. Further, the VHs and VLs may be subjected to DNA shuffling to select for improved biophysical properties.

FIELD OF THE INVENTION

This is a national phase entry application claiming the benefit of PCTApplication No. PCT/CA2006/000451 filed Mar. 24, 2006, which claimspriority to U.S. Provisional Patent Application No. 60/664,954 filedMar. 25, 2005, now ABN.

A sequence listing in text file format entitled 2010 11 26corrected—sequence—listing, created on Nov. 26, 2010, and having a sizeof 54kB is incorporated herein by reference.

This invention relates to the isolation, identification and manipulationof polypeptides, especially monomeric human antibody fragments.

BACKGROUND OF THE INVENTION

Antibodies in vertebrates are typically composed of paired heavy (H) andlight (L) chains. The first domain of the combined H and L chains, theV_(H) and V_(L), are more variable in sequence, and this is the portionof the antibody that recognizes and binds to the antigen. The V_(H) andV_(L) domains recognize the antigen as a pair.

The immune repertoire of camelidae (camels, dromedaries and llamas) isunique in that it possesses unusual types of antibodies referred to asheavy-chain antibodies (Hamers, Casterman C. et al., 1993). Theseantibodies lack light chains and thus their combining sites consist ofone domain, termed V_(H)H.

Recombinant V_(H)H single-domain antibodies (sdAbs) provide severaladvantages over single-chain Fv (scFv) fragments derived fromconventional four-chain antibodies. While sdAbs are comparable to theirscFv counterparts in terms of affinity, they outperform scFvs in termsof solubility, stability, resistance to aggregation, refoldability,expression yield, and ease of DNA manipulation, library construction and3-D structural determinations. Many of the aforementioned properties ofV_(H)H sdAbs are desired in applications involving antibodies.

However, the non-human nature of V_(H)Hs limits their use in humanimmunotherapy due to immunogenicity. In this respect, human V_(H) andV_(L) sdAbs are ideal candidates for immunotherapy applications becausethey are expected to be least immunogenic.

Human V_(H)s and V_(L)s, however, are by and large prone to aggregation,a characteristic common to V_(H)s and V_(L)s derived from conventionalantibodies (Davies, J. et al., 1994; Tanha, J. et al., 2001; Ward, E. S.et al., 1989). Thus, attempts have been made to obtain monomer humanV_(H)s and V_(L)s suitable for antibody applications. Such V_(H)s andV_(L)s have also displayed other useful properties typical of V_(H)Hssuch as high expression yield, high refoldability and resistance toaggregation. Synthetic libraries built on these V_(H)s and V_(L)s aslibrary scaffolds might serve as a promising source of therapeuticproteins.

Camelization as well as lamination which involves incorporating keysolubility residues from camel and llama V_(H)HS, respectively, intohuman V_(H)s or V_(L)s have been employed to generate monomeric humanV_(H)s and V_(L)s. Synthetic sdAb libraries constructed based on theseV_(H)s and V_(L)s and generated by CDR randomization were shown to befunctional in terms of yielding binders to various antigens (Davies, J.et al., 1995; Tanha, J. et al., 2001).

In another approach, fully human monomeric V_(H)s and V_(L)s wereisolated from human synthetic V_(H) and V_(L) libraries withoutresorting to engineering of the sort mentioned above. In one experimenta monomeric human V_(H), was discovered when a human V_(H) library waspanned against hen egg lysozyme (Jespers, L. et al., 2004b). Morerecently, a selection method based on reversible unfolding and affinitycriteria yielded many monomeric V_(H)s from synthetic human V_(H)libraries (Jespers, L. et al., 2004a). This finding underlined the factthat an appropriate selection method is key to efficient capturing ofrare monomer human V_(H)s with desirable biophysical properties.

OBJECTS OF THE INVENTION

A first object of the invention is to provide a high throughputscreening method for identifying polypeptides, especially antibodyfragments, with improved biophysical properties, including solubility,high expression, and/or stability (such as high refolding after thermaldenaturation, high resistance to chemical denaturant, and highresistance to proteases, in particular gastrointestinal proteases suchas trypsin).

A second object of the invention is to provide a high throughputscreening method for identifying monomeric human V_(H)s and V_(L)s.

A third object of the invention is to identify, isolate and characterizemonomeric human V_(H)s and V_(L)s.

A fourth object of the invention is to construct and characterizemultimers of antibody fragments, especially monomeric human V_(H)s andV_(L)s.

A fifth object of the invention is to construct display libraries frompolypeptides, especially antibody fragments, and most especiallymonomeric human V_(H)s and V_(L)s.

A sixth object of the invention is to provide a DNA shuffling method forproducing polypeptides, especially antibody fragments, and mostespecially monomeric human V_(H)s and V_(L)s with improved biophysicalproperties.

SUMMARY OF THE INVENTION

A method is provided for isolating polypeptides, preferably antibodyfragments, and most preferably human V_(H)s and V_(L)s with desirablebiophysical properties (solubility, stability, high expression,monomericity, non-aggregation, binding specificity). The method includesthe steps of obtaining a phage display library capable of expressing avariety of polypeptide sequences, allowing infection of a bacterial lawnby the library phage, and identifying phage which form larger thanaverage plaques on the bacterial lawn. The phage are then isolated, andsteps are taken to sequence or otherwise characterize the polypeptidesequences.

The invention also provides for polypeptides, especially monomeric humanV_(H)s and V_(L)s, identified by the above method, which may be usefulfor immunotherapy, and/or as diagnostic or detection agents. Themonomeric human V_(H)s and V_(L)s may also be combined to form dimers,trimers, pentamers or other multimers, which may be useful forimmunotherapy and/or as diagnostic or detection agents.

The polypeptides identified by the above method, including human V_(H)sand V_(L)s, can be manipulated by methods such as DNA shuffling toselect for improved biophysical properties such as solubility,stability, monomericity, high expressibility, binding specificity andhuman origin.

The polypeptides identified by the above method, including human V_(H)sand V_(L)s, may also be used to generate further display libraries,which can then in turn be used to isolate further polypeptides by theabove method.

In a first aspect, the present invention provides a method ofidentifying target polypeptides, comprising a) obtaining a phage displaylibrary capable of expressing a variety of polypeptide sequences, b)allowing infection of a bacterial lawn by the library phage and c)identifying phage which form larger than average plaques on thebacterial lawn.

In a second aspect, the present invention provides polypeptide having anamino acid sequence selected from the group consisting of: SEQ IDNO:8-54

In a third aspect, the present invention provides a V_(H) antibodyfragment comprising at least one amino acid sequence selected from thegroup consisting of: SEQ ID NO:8-22.

In a fourth aspect, the present invention provides a V_(L) antibodyfragment comprising at least one amino acid sequence selected from thegroup consisting of: SEQ ID NO:23-54.

In a fifth aspect, the present invention provides A method for producingpolypeptides with desirable biophysical properties, comprising the stepsof a) providing at least one first nucleic acid sequence that encodes anantibody fragment comprising at least one amino acid sequence selectedfrom SEQ ID NOS. 8-54 or that encodes a polypeptide sequence having anamino acid sequence selected from SEQ ID NOS. 8-54 or the CDR1, CDR2 orCDR3 portion thereof and having a first desirable property; b) providingat least one second nucleic acid sequence that encodes an antibodyfragment having a second desirable property; c) cutting the at least onefirst and at least one second nucleic acid sequences into randomfragments; d) reassembling the random fragments; e) expressing therandom fragments; and f) screening the expressed random fragments forthe first and second desirable properties.

DETAILED DESCRIPTION OF THE DRAWINGS

Figure Legends

FIG. 1. A pictorial representation of selected example results: Thecontrast in plaque size between phages displaying a soluble V_(H)(HVHP428) and those displaying an insoluble one (BT32/A6). The photodepicts a part of the bacterial lawn agar plate which was magnified toenhance plaque visualization. Although the plate contained an equalnumber of each of the two plaque types, the photo essentially containsthe large, HVHP428 plaques. The majority of the BT32/A6 plaques were toosmall to produce clear, well-defined images in the photo. The plaquesmarked by arrows, thus, represent a minor proportion of BT32/A6 phageswhich were large enough to be visible in this image. Asterisks marksrepresentative plaque sizes for HVHP428 phages. The identities ofplaques were determined by DNA sequencing.

FIG. 2. Amino acid sequence of human V_(H)S selected based on affinityfor protein A and plaque size. (SEQ ID NOS.: 8-22 disclosed respectivelyin order of appearance). The dots in the sequence entries indicate aminoacid identity with HVHP2M10 or HVHP44. Dashes are included for sequencealignment. Residues at the key solubility positions and residue 57Twhich associates with V_(H)s/V_(H)Hs with protein A binding property arein bold. The Kabat numbering system is used. The total “frequency” valueis 114. CDR=complementarity determining region; FR−framework region; glnseq=germline sequence. CDR=complementarity determining region;FR=framework region; gin seq=germline sequence

FIG. 3. Aggregation tendencies of the human V_(H)s. Gel filtrationchromatograms comparing the oligomerization state of a human V_(H)isolated in this study (HVHP428) to that of a llama V_(H)H (H11C7) and atypical human V_(H) (BT32/A6). The peak eluting last in eachchromatogram corresponds to monomeric V_(H). The dimeric H11C7 peak ismarked by an arrow. B, One-dimensional ¹H NMR spectra of HVHP414 at 800MHz (i), HVHP423 at 500 MHz (ii) and HVHP428 at 800 MHz (iii). Thespectra in the left panel are scaled up by a factor of two to enablebetter viewing of low-intensity signals.

FIG. 4. Stability of the human V_(H)S in terms of their resistance totrypsin at 37° C. and integrity following long incubation at 37° C. A,SDS-PAGE comparing the mobilities of the untreated and trypsin-treatedHVHP414 V_(H) at 15, 30 and 60 min relative to a 21 kDa marker.HVHP414-cMyc denotes HVHP414 V_(H) lacking the c-Myc. B, Molecular massprofiles obtained by mass spectrometry of untreated and trypsin-treated(60 min) HVHP414 V_(H) (SEQ ID NO.: 18). The mass spectrometry profileof the treated V_(H) is superimposed onto that for the untreated one toprovide a better visual comparison. The experimental molecular mass ofthe untreated V_(H) is 14,967.6 Da, which is essentially identical tothe expected molecular mass, 14,967.7 Da. The observed molecular mass ofthe trypsin-treated V_(H) (13,368.5 Da) indicates loss of 13 amino acidsat the C-terminus by cleavage at K (Lys) in the c-Myc tag to give anexpected molecular mass of 13,368.0 Da. The trypsin cleavage site isshown by a vertical arrow above the amino acids sequence of HVHP414. C,Gel filtration chromatograms comparing the oligomerization state of the37° C-treated HVHP420 V_(H) (upper profile) to that of untreated V_(H)(lower profile). The chromatograms were shifted vertically because theywere indistinguisable when superimposed. The major and minor peaks ineach chromatogram correspond to monomeric and dimeric V_(H)s,respectively. The dimeric V_(H) constitutes 3% of the total protein. Theinset shows the sensorgram overlays for the binding of 37° C-treatedHVHP420 to protein A at various concentrations. The V_(H)s used fortemperature stability studies were from stocks which had already been at4° C. for several months.

FIG. 5. Sensogram overlays showing the binding of native (thick lines)and refolded (thin lines) HVHP423 to immobilized protein A at 75, 100,150 and 200 nM concentrations. K_(D)n and K_(D)ref were calculated fromrespective sensograms and used to determine RE as described below.

FIG. 6. Amino acid sequences of the human V_(L)s selected based onaffinity for protein L and plaque size (SEQ ID NOS.: 23-54 disclosedrespectively in order of appearance). The dots in the sequence entriesindicate amino acid identity with HVLP333. Dashes are included forsequence alignment. See the V BASE for sequence numbering and CDRdesignation. L6, A27, L2, L16, 02/012, A30 and 1b are V germlinedesignation. J germline designations are in the brackets. NE, not found.

FIG. 7. Size exclusion chromatograms of human V_(L) domains. In A, theV_(L)s were applied at a concentration of 0.6 mg/ml. In B, the V_(L)swere applied at their highest concentration available: HVLP342, 1.0mg/ml; HVLP3103, 5.9 mg/ml; HVLP335, 4.9 mg/ml; HVLP351, 0.89 mg/ml. “#”and “*” represent aggregate and monomer peaks, respectively. Theaggregates elute in the exclusion volume. The peak marked by an arrow inthe HVLP342 panel (B) is the carry over from a previous run.

FIG. 8. Sensorgram overlays showing the binding of V_(L)s to immobilizedprotein L at concentrations of 0.2, 0.5, 0.75, 1, 2, 3, 5 and 10 μM(HVLP389, HVLP351 and HVLP364); 1, 2, 3, 5, 7.5 and 10 nM (HVLP342);0.2, 0.5, 1, 2, 3, 5 and 10 μM (HVLP335); 0.2, 0.5, 1, 1.5, 2 and 5 μM(HVLP325), 0.2, 0.5, 0.75, 1, 1.5, 2, 3 and 5 μM (HVLP3103) and 1, 2, 3,4, 5 and 6 nM (HVLP324). The sensorgrams for HVLP324 and HVLP342bindings to the low affinity site of protein L are not included but thecalculated K_(D)s are recorded in Table 3.

FIG. 9. Bindings of HVHP328PTV2 to protein A and HVLP335 PTV2 to proteinL in surface plasmon resonance experiments. (A) Sensorgram overlaysshowing the binding of HVH28PTV2 to immobilized protein A at 1, 2, 3, 4,6, 8 and 10 nM concentrations. (B) Sensorgram overlays showing thebinding of HVLP335PTV2 to immobilized protein L at 1, 2, 2.5, 3, 3.5, 4and 4.5 nM concentrations. The binding data are recorded in Table 4.

FIG. 10. Figure showing the results of the microagglutinationexperiments with S. aureus cells. The concentration of the pentamersdecreases two-fold from well 1 to well 11 with well 12 having thepentamers replaced with PBS buffer. The top row wells containHVHP328PTV2 pentamer and the bottom ones HVLP335PTV2 pentamer. Theconcentrations of the pentamers in wells 1 to 6 are 215, 108, 54, 27, 13and 7 μg/ml, respectively.

DETAILED DESCRIPTION OF THE INVENTION

It is desirable to identify polypeptides, especially antibody fragments,that are of human origin, soluble, stable, resistant to aggregation,refoldable, highly expressed, easily manipulated at the DNA level, idealfor library construction and for 3-D structural determinations. Suchantibody fragments are useful for a wide variety of immunotherapeuticalapplications, and also as diagnostic and detection agents. Humanmonomeric V_(H) and V_(L) antibodies are of particular interest, as theyare likely to have many of the above-mentioned properties.

Polypeptides with the above-mentioned properties may be identified byhigh throughput screening of libraries capable of expressing a varietyof polypeptide sequences. For example, phage display libraries(preferably filamentous phage such as M13 or fd) may be screened byinfecting a field of bacteria susceptible to the phage (a bacteriallawn) with the phage, then determining which phages have successfullylysed the bacteria by looking for clear, bacteria-free areas known asplaques. Phages displaying monomeric laminated V_(H)s and V_(L)s formlarger plaques on bacterial lawns than phages displaying fully humanV_(H)s with aggregation tendencies. Thus, plaque size may be used as ameans of identifying rare, naturally-occurring monomer V_(H)s and V_(L)sfrom the human V_(H) repertoire.

The method disclosed herein is also useful in identifying soluble,stable (stability covers a number of characteristics, including but notlimited to high thermal refolding efficiency, high melting temperature,maintaining functionality after long (several days) incubation at 37°C., resistant to chemical denaturants, resistant to proteases, having along shelf life at below 0° C., and 4° C., and at room temperature,maintaining functionality in intracellular environments, and maintainingfunctionality inside the human body, such as in the bloodstream) andhigh expressing proteins of differing origins, including:

-   1. V_(H)s, V_(L)s, Fabs, scFvs and whole antibodies such as IgGs,    more specifically human ones-   2. Protein variants based on non-antibody scaffolds single-chain    T-cell receptors, T-cell receptor domains, transferin, lipocalins,    kunitz domains, ankyrin repeats, and cytotoxic    T-lymphocyte-associated antigen (CTLA-4), including human ones-   3. Vaccines such as viral and bacterial protein vaccines-   4. Therapeutic proteins, e.g., insulin, growth hormone,    arythropoietin

5. Proteinacious diagnostic and biochemical reagents, e.g., protein A,protein G.

Once polypeptides have been identified by this method, they can be usedto construct additional libraries. This is done by selecting a nucleicacid sequence of, for example, a VH. Oligonucleotides with randomizedcodons are created and incorporated into the VH sequence. Thus, eachunique oligonucleotide is incorporated into a VH gene, and the modifiedVH genes constitute a library of sequences with slight variations.Typically, the oligonucleotides are designed such that the CDRs or loopsof the VH are randomized. For example, one, two or all three of VH CDRsmay be randomized. The VH library is then cloned into an appropriatevector, depending on the type of library to be used, and the nucleicacid sequences are expressed as polypeptides. The library is screenedfor molecules that bind to the library polypeptides, typically bypanning. The libraries may be phage display libraries, or other displaylibraries such as ribosome display and yeast display.

Polypeptides identified by the method discussed herein may be used forimmunotherapy by, for example, the cross-linking of monomers to formdimers, trimers, pentamers and other multimers. This may result inbetter affinity for antigen molecules and slower dissociation rates forsome antigens. Another possible approach is to link or fuse polypeptidesto a variety of molecules with various functions. For example, antibodyfragments may be linked to radionuclides, cytotoxic drugs, toxins,peptides, proteins, enzymes, liposomes, lipids, T-cell superantigens orviruses in order to target and destroy or modify specific cells ormolecules.

Once the V_(H)s or V_(L)s identified by the selection method describedherein have been isolated, they can be further manipulated to select forimproved biophysical properties such as solubility, stability,monomericity, binding specificity, human origin or high expressability.This can be achieved by in vitro recombination techniques such as DNAshuffling or a staggered extension process. DNA shuffling involvescutting the nucleic acid sequence of first (donor) and second (acceptor)polypeptides, such as antibody fragments, into random fragments, thenreassembling the random fragments by a PCR-like reaction. Thereassembled fragments are then screened to select for the desiredproperties.

For example, one or more VHs with high stability (donors) can be mixedwith one or more VHs lacking sufficient stability (acceptors) andsubjected to DNA shuffling. This generates mutants of the acceptor VHswhich have incorporated stability residues from the donor VHs. The newlystable mutants can be identified by the methods described herein, orthrough other evolutionary protein screening systems such as ribosomedisplay, yeast display, bacterial cell display and phage display.Similarly, this technique can be used to transfer desirable traits suchas solubility, monomericity, and high expression.

This technique may be used where both donor and acceptor V_(H)s havedesirable properties, to produce a V_(H) with both properties. Forexample, an unstable donor V_(H) which binds to an important therapeuticor diagnostic ligand can be shuffled with a stable acceptor V_(H). Inorder to ensure that new generated stable V_(H)s also have the abilityto bind to the ligand, the screening system may involve a ligand bindingstep.

DNA shuffling may also be useful for humanizing non-human V_(H)s such ascamelid heavy chain antibody variable domains and nurse shark andwobbegong shark variable domains, or non-human V_(L)s which bind totherapeutic targets. Human V_(H)s and V_(L)s with desirable propertiessuch as solubility, stability, monomericity and high expressability maybe used as donors. For example, one or more human V_(H)s with goodstability (donors) can be mixed with one or more non-human therapeuticV_(H)s (acceptors) and subjected to DNA shuffling. This generatesmutants of the acceptor V_(H)s which are both stable and humanized. Thenewly generated humanized and stable mutants can be identified by themethods described herein, or through other evolutionary proteinscreening systems such as ribosome display, yeast display, bacterialcell display and phage display. In a further example, the acceptor V_(H)could be a therapeutic V_(H)H (camelid heavy chain antibody variabledomain).

Further, this technique is also useful for selecting desirableproperties of polypeptides other than V_(H)s and V_(L)s. As discussedabove, the donor polypeptide and the acceptor polypeptide may be bothhuman, or the donor may be human and the acceptor non-human.

A possible approach for imparting solubility, monomericity, highexpressability or stability to V_(H)s and V_(L)s may be through graftingcomplementarity determining regions (CDRS) onto acceptor V_(H)s andV_(L)s. Since CDRs are known to be involved in the solubility andstability of single-domain antibodies, and accordingly the grafting ofthese regions, such as the CDRs from V_(H)s and V_(L)s isolated by themethods described herein, may impart solubility and/or stability toacceptor V_(H)s and V_(L)s.

Human Monomeric V_(H)s and V_(L)s

Several monomeric human V_(H)s with different germline and overallsequences were identified (see FIG. 1 and SEQ ID NO. 8 through 22) froma naïve human V_(H) phage display library by this selection method basedon phage plaque size. The V_(H)s remain functional and monomericfollowing trypsin treatment at 37° C., weeks of incubations at 37° C. ormonths of storage at 4° C., have high thermal refolding efficiencies,are produced in good yields in E. coli and possess protein A bindingactivity.

In addition, several monomeric human V_(L)s were identified (see FIG. 6and SEQ ID NO. 23 through 54). The V_(L)s are also produced in goodyields in E. coli and possess protein L binding activity.

Such properties will also be manifested by V_(H)s from syntheticlibraries that utilize the above V_(H)s as scaffolds. Thus, suchlibraries may yield therapeutic or diagnostic V_(H)s which would havegood efficacy at physiological temperature, extended shelf life and acost-effective production. High thermal refolding efficiencycharacteristic would further extend the biotechnological applications ofthese libraries to situations where V_(H) binders are required tomaintain their activity after exposure to transient high temperatures.The V_(H)s should also be very suitable for intrabody applicationsbecause of their desirable biophysical properties. The protein A bindingproperty will simplify V_(H) purification and detection in diagnostictests, immunoblotting and immunocytochemistry and can be exploited toenhance library performance by removing nonfunctional V_(H)s from thelibraries. Similarly, libraries that utilize V_(L)s as scaffolds willyield therapeutic or diagnostic V_(L)s which have similarly desirableproperties. Since V_(L)s bind with protein L, V_(L) purification anddetection is simplified by taking advantage of this protein L bindingproperty.

Display libraries built on the present V_(H)s and V_(L)s may also be auseful source of diagnostics and detection agents.

Previously reported fully human V_(H)s with favorable biophysicalproperties were based on a single V germline sequence: DP47 ((Jespers,L. et al., 2004b; Jespers, L. et al., 2004a). The observation that themonomeric human V_(H)s in this study stem from six different germlinesequences including DP-47, demonstrates that stable V_(H)s are notrestricted in terms of germline gene usage. In fact, it is very likelythat we would have isolated monomeric V_(H)s of family and germlineorigins different from the ones we describe here had we not restrictedour selection to a subset of V_(H)3 family V_(H)s with protein A bindingactivity. It is not possible to pinpoint amino acid mutations (Table 1)responsible for the observed biophysical behavior of the present V_(H)sdue to the occurrence of multiple mutations in V_(H)s and the fact thatCDR3 is also known to be involved in shaping the biophysical profiles ofsdAbs. It may be, however, that mutations at positions known to beimportant for sdAbs stability and solubility, e.g., V37F in HVHP423 andHVHP44B, or mutations occurring multiple times at the same position,e.g., L5V/Q and V5Q in nine V_(H)s, have a role in determining V_(H)sbiophysical properties. In terms of library construction, it would bedesirable that the monomericity of the present V_(H)s not be dependenton CDRs, in particular CDR3, so that CDR randomization be performedwithout the worry of jeopardizing library stability. In this regard, theV_(H)s with smaller CDR3, e.g., HVHB82, may be preferred scaffolds sincethere would be less dependence on CDR3 for stability.

The diversity of the present V_(H)s and V_(L)s in terms of overallsequence and CDR3 length should allow the construction ofbetter-performing libraries. Synthetic V_(H) libraries have beenconstructed on single scaffolds. Such an approach to repertoiregeneration is in sharp contrast to the natural, in vivo “approach” whichutilizes a multiplicity of scaffolds. Based on the sequences reportedhere one can take advantage of the availability of the diverse set ofV_(H)s and V_(L)s and create libraries which are based on multiple V_(H)and V_(L) scaffolds. Such libraries would be a better emulation of invivo repertoires and therefore, would have a more optimal complexity. Ofthe three CDRs in sdAbs, CDR3 generally contributes most significantlyto repertoire diversity and for this reason CDR3 randomization on V_(H)and V_(L) scaffolds are typically accompanied by concomitant varying ofCDR3 length. While this significantly improves library complexity, itmay also compromise library stability by disrupting the length of theparental scaffold CDR3. The heterogeneity of the V_(H)s and V_(L)sdisclosed herein in terms of CDR3 length permit the creation oflibraries with both good complexity, good stability and good biophysicalcharacteristics. Such libraries would preferably consist ofsub-libraries, where each sub-library is created by CDR3 randomization(and CDR1 and/or CDR2 randomization, if desired) on a single V_(H) orV_(L) scaffold without disrupting the parental CDR3 length.

The versatility of the present V_(H)s and V_(L)s is also beneficial interms of choosing an optimal V_(H) or V_(L) framework for humanizingV_(H)Hs, V_(H)s and V_(L)s which are specific to therapeutic targets.High affinity camelid V_(H)Hs against therapeutic targets can beobtained from immune, non-immunized or synthetic V_(H)H libraries withrelative ease and be subsequently subjected to humanization (CDRgrafting, resurfacing, deimmunization) to remove possible V_(H)Himmunogenicity, hence providing an alternative to human V_(H) libraryapproach for production of therapeutic V_(H)s. Generating high affinitytherapeutic V_(H)s by the latter approach may often require additionaltedious and time consuming in vitro affinity maturation of the leadbinder(s) selected from the primary synthetic human V_(H) libraries.

Nonhuman V_(H)s against therapeutic targets can be obtained from immune,non-immunized or synthetic V_(H) libraries with relative ease and besubsequently subjected to humanization (CDR grafting, resurfacing,deimmunization) to eliminate nonhuman V_(H) immunogenicity, henceproviding an alternative to human V_(H) library approach for productionof therapeutic V_(H)s.

Nonhuman V_(L)s against therapeutic targets can be obtained from immune,non-immunized or synthetic V_(H)H libraries with relative ease and besubsequently subjected to humanization (CDR grafting, resurfacing,deimmunization) to eliminate V_(H)H immunogenicity, hence providing analternative to human V_(L) library approach for production oftherapeutic V_(L)s.

A number of evolutionary approaches for selection of proteins withimproved biophysical properties have been described (Forrer, P. et al.,1999; Waldo, G. S., 2003); (Jespers, L. et al., 2004a; Jung, S. et al.,1999; Matsuura, T. et al., 2003). Typically, stability pressure isrequired to ensure preferential selection of stable variants overunstable or less stable ones from a library population. For example, ina related work, heat treatment of V_(H) phage display libraries wasrequired to select aggregation resistant V_(H)s (Jespers, L. et al.,2004a). Examples of evolutionary selection approaches involving phagedisplay include conventional phage display, selectively infective phageand the proteolysis approaches. In the first two approaches affinityselection is used to select stable species from a library, based on theassumption that stable proteins possess better binding properties fortheir ligand than the unstable ones. However, even with the additionalinclusion of a stability selection step, these approaches may primarilyenrich for higher affinity rather than for higher stability (Jung, S. etal., 1999). A binding step requirement also limits the applicability ofthese approaches to proteins with known ligands. The third, proteolysisapproach is based on the fact that stable proteins are generally compactand therefore are resistant to proteases whereas the unstable ones arenot. The phage display format is engineered in such a way that theprotease stability of the displayed protein translates to phageinfectivity. Thus, when a variant phage display library is treated witha protease, only the phages displaying stable proteins retain theirinfectivity and can subsequently be selected by infecting an E. colihost. Since this approach is independent of ligand binding, it hasgeneral utility. However, even stable and well folded proteins haveprotease sensitive sites, e.g., loops and linkers, and this couldsometimes hinder the selection of stable species in a proteolysisapproach (Bai, Y. et al., 2004).

By contrast, in the present evolutionary approach, proteins withsuperior biophysical properties are simply identified by the naked eye.The approach does not require ligand binding, proteolysis ordestabilization steps, and thus, avoids complications which may beencountered in the reported selection approaches. No requirement for abinding step also means that this approach has general utility. As anoption, a binding step may be included to ensure that the selectedproteins are functional. However, the dependency of the present approachon plating (for plaque visualization) introduces a possible logisticallimitation in terms of the number of plates that can be handled and thuslimits its application to smaller libraries. Nonetheless, the utility ofthe current approach can be extended to large libraries, if the libraryis first reduced to a manageable size. This can be done, for example, byincorporating into the selection system a step which would remove largepopulations of unstable species, e.g., library adsorption on a protein Asurface, or on a hydrophobic interaction column to remove poorly foldedproteins with exposed hydrophobic surfaces (Matsuura, T. et al., 2003).Here, the approach was used to select V_(H)s and V_(L)s of goodbiophysical properties in a background of very unstable V_(H)s andV_(L)s. However, it may be more difficult to select the “best” speciesfrom a mutant library which is populated with proteins with reasonablygood stabilities. In this case, the lead variants may be identifiedbased on the rate of plaque formation by using shorter incubation times,or based on plaque size and frequency criteria.

The present selection approach can be extended to identification ofstable and well-folded antibody fragments such as scFvs and Fabs withthe optional inclusion, in the selection system, of a binding stepinvolving protein L, A or any ligand, as well as stable non-antibodyscaffolds and variants thereof. Moreover, the observed correlationbetween phage plaque size and V_(H) expression yield means that one canutilize the present approach for acquiring high-expressing versions ofproteins with otherwise poor or unsatisfactory expression from mutantphage display libraries. This application would be particularlyappealing in the case of therapeutic proteins or expensivepoor-expressing protein reagents where boosting protein expression wouldsignificantly offset protein production cost.

Binding Analyses of Pentamers

Both V_(L)s and V_(H)S are amenable to pentamerization and thepentamerization can be used to quickly convert a low affinity V_(L) orV_(H) monomer to a high affinity V_(L) or V_(H) pentamer. Such pentamersare invaluable diagnostics and detection agents. In such applications,the binding of a V_(L) or V_(H) pentamer to its target can be detectedby a reporter molecule such as an enzyme (for example, horse radishperoxidase or alkaline phosphatatase), or a fluorescent moleculeconjugated to the pentamer. Alternatively, the binding of the pentamercan be detected by a secondary molecule which is conjugated to areporter molecule. The secondary molecule can be specific to thepentamer itself or to a tag thereof, such as 6His tag (SEQ ID NO.: 55)or c-Myc tag. For example, a typical secondary molecule is animmonoglobulin.

The interactions between the V_(H)s and protein A and V_(L)s withprotein L are fundamentally different from those between V_(H)s andV_(L)s with their target antigens. The antigen binding of a V_(H) or aV_(L) involves three antigen binding loops which form the combining siteof an antibody domain. The protein A binding of a V_(H) with protein Abinding activity and a V_(L) with protein L binding activity involvebinding sites and residues on the antibody domains that are totallydistinct from the antibody combining site. Thus, a V_(H) with protein Abinding activity can simultaneously bind to protein A and its targetantigen and a V_(L) with protein L binding activity can simultaneouslybind to protein L and its target antigen. Since the present V_(H)s andV_(L)s have affinity for protein A and L, respectively, protein A and Lcan be used as the secondary molecule for detection and diagnosticapplications mentioned above. The human V_(H) and V_(L) pentamers canalso be used for therapy.

Pathogen Detection by the Pentamers

The protein A and L binding activity of the V_(H)s and V_(L)s can beused to detect bacteria which have protein A and/or L on their surfaces.Protein A is present on the surface of the pathogenic bacteria,Staphylococcus aureus. Thus, the V_(H)s with protein A binding activitysuch as the ones described here can be used to detect S. aureus.Similarly, the V_(L) monomers and V_(L) pentamers with protein L bindingactivity can be used for the detection of bacteria, in particularpathogenic bacteria such as Peptostreptococcus magnus, which haveprotein L on their cell surface.

Protein L is implicated as a virulent factor in the pathogenesis of P.magnus (Ricci, S. et al., 2001) in humans. In vaginosis, protein L isthought to exert its effect by cross-linking surface associated IgE.V_(L) monomers and/or pentamers with protein L binding activity havepotential as therapeutics since they could interfere with the IgEcross-linking action of protein L.

Protein A is implicated as a virulent factor in the pathogenesis of S.aureus in humans (Fournier, B. et al., 2004). Its virulence has beenattributed to its ability to interact with host components includingbinding to antibodies. V_(H) monomers and/or pentamers with protein Abinding activity have potential as therapeutics since they couldinterfere with the interaction of protein A with host components.

EXAMPLES

Identification and Sequence Analysis of Monomeric Human V_(H)s

During the course of the construction of fully human and laminated humanV_(H) libraries, it was learned that the phages displaying monomericlaminated V_(H)s formed larger plaques on bacterial lawns than phagesdisplaying fully human V_(H)s with aggregation tendencies. Thus, plaquesize was used as a means of identifying rare, naturally-occurringmonomer V_(H)s from the human V_(H) repertoire (FIG. 1). To this end, aphage library displaying human V_(H)s with a size of 6×10⁸ wasconstructed and propagated as plaques on agar plates. On the titerplates, the library consisted essentially of small plaques interspersedwith some large ones. PCR on twenty clones revealed that the smallplaques corresponded to the V_(H)-displaying phages while the large onesrepresented the wild type phages, i.e., phages lacking V_(H) sequenceinserts. None of the V_(H)-displaying phages were found with largeplaque morphology. This was not unexpected due to the paucity of themonomeric V_(H)s in the human repertoire and the large size of thelibrary. To facilitate the identification of monomeric V_(H)s, it wasdecided to reduce the library to a manageable size and remove theinterfering wild type phages with large-plaque-size morphology bypanning the library against protein A which binds to a subset of humanV_(H)s from V_(H)3 family.

Following a few rounds of panning, the library became enriched for phageproducing large plaques, and PCR and sequencing of more than 110 suchplaques showed that all had complete V_(H) open reading frames. The sizeof the large plaques which were picked for analysis is represented inFIG. 1. Sequencing revealed fifteen different V_(H)s which belonged tothe V_(H)3 family and utilized DP-38, DP-47, V349, V3-53, YAC-5 or 8-1Bgermline V segments (Table 1; FIG. 2). DP-38 and DP47 germline sequenceshave been previously implicated in protein A binding. In addition, allV_(H)s had a Thr residue at position 57 (FIG. 2), consistent with theirprotein A binding activity. The most frequently-utilized germine Vsegment was DP-47 which occurred in over 50% of the V_(H)s, but the mostfrequent clone (i.e., HVHP428; relative frequency 46%) utilized theV3-49 germline V segment. HVHP429 with a DP-47 germline sequence was thesecond most abundant V_(H) with a relative frequency of 21% (FIG. 2).The V_(H) CDR3 lengths ranged from 4 amino acids for HVHB82 to 16 aminoacids for HVHP430 amino acids, with HVHP430 having a pair of Cysresidues in CDR3. Amino acid mutations with respect to the parentalgermline V segment (residues 1-94) and FR4 (residues 103-113) sequences,were observed in all V_(H)s and ranged from two mutations for HVHP44(L5V and Q105R) and HVHB82 (E1Q and L5Q) to sixteen mutations forHVHP426 (Table 1). Mutations were concentrated in the V segments; onlytwo mutations were detected in all the fifteen FR4s, at positions 105and 108. HVHP44 and HVHB82 differed from other V_(H)s in that they bothhad a positively-charged amino acid at position 105 instead of a Gln(Table 1; FIG. 2). However, while the positively-charged amino acid inHVHP44 was acquired by mutation, the one in HVHB82 was germline-encoded.Except for HVHP423 and HVHP44B, the remaining V_(H)s had the germlineresidues at the key solubility positions: 37V/44G/45L/47W or37F/44G/45/L47W (HVHP428); HVHP423 and HVHP44B had a V37F mutation.Mutations at other positions which are shown or hypothesized to beimportant in V_(H) solubility included seven E6Q, three S35T/H, one R83Gand one K83R, one A84P and one T84A and one M108L. Frequent mutationswere also observed at positions 1 and 5 which included eleven E1Q, eightL5V/Q and one V5Q mutations.

Biophysical Characterization of the Human V_(H)s

All V_(H)s except HVHP44B, which was essentially the same as HVHP423,were expressed in one-liter-culture volumes in E. coli strain TG1 infusion with c-Myc-His₅ tag and purified to homogeneity from periplasmicextracts by immobilized metal affinity chromatography (IMAC). Theexpression yields ranged from 1.8 to 62.1 mg of purified protein perliter of bacterial culture in shaker flasks with majority of V_(H)shaving yields in several milligrams (Table 2). In the instance ofHVHP423 and HVHP430, another trial under “apparently” the sameexpression conditions gave yields of 2.4 and 6.4 mg as opposed to 62.1and 23.7 mg, respectively. This implies that for many of the V_(H)sdescribed here optimal expression conditions should be achieved, withoutmuch effort, resulting in expression yields significantly higher thanthe reported values in Table 2. As expected, all the V_(H)s bound toprotein A in surface plasmon resonance (SPR) analyses, with K_(D)s of0.2-3 μM, a range and magnitude comparable to the ones reportedpreviously for llama V_(H)H variants with protein A binding activity.None of the VHs bound to the Fab reference surface.

The aggregation tendency of the human V_(H)s was assessed in terms oftheir oligomerization states by gel filtration chromatography and NMR(Table 2). All V_(H)s were subjected to Superdex 75 gel filtrationchromatography. Similar to a llama V_(H)H, i.e., H11C7, all V_(H)s gavea symmetric single peak at the elution volume expected for a monomer,and were substantially free of any aggregates (see the example forHVHP428 in FIG. 3A. In contrast, a typical human V_(H) (i.e., BT32/A6)formed considerable amount of aggregates. For three of the V_(H)s, aminor peak with a mobility expected for a V_(H) dimer was also observed.SPR analyses of the minor peaks gave off-rate values which weresignificantly slower than those for the monomer V_(H)s, consistent withthem being dimers. The dimer peak was also observed in the case of thellama V_(H)H, H11C7. The folding and oligomerization states of theV_(H)s at high concentrations were further studied by NMR spectroscopy.As shown in Table II, all the V_(H) proteins studied appeared to berelatively soluble and assumed a well-folded three-dimensionalstructure. One-dimensional NMR spectra of the V_(H) fragments (FIG. 3B)showed structure folds characteristic of V_(H) domains. The state ofprotein aggregation was also assessed by use of an PFG-NMR diffusionexperiment for the HVHP414 fragment and two isoforms, VH14 and VH14-cMycwith and without the c-Myc sequence, of the HVHP414. VH14 is a modifiedversion of HVHP414 with a c-Myc N132E mutation and with an additionalmethionine residue at the N-terminus. In brief, the PFG-NMR data (notshown) indicated that all the protein samples had expected monomericmolecular weights even at the relatively high protein concentrationsused for NMR experiments.

The stability of the V_(H)s was further investigated in terms of theirresistance to trypsin at 37° C. integrity following long incubations at37° C. Trypsin cleaves polypeptide amide backbones at the C-terminus ofan Arg or a Lys residue. There are 9-13 Arg and Lys residues in thehuman V_(H)s (FIG. 2). There is also an additional Lys residue in theC-terminal c-Myc tag which is susceptible to digestion by trypsin. FIG.4 a is an SDS-PAGE analysis of HVHP414 during trypsin digestion. Within1 h the original band was completely converted to a single product whichhad a mobility expected for the V_(H) with no c-Myc-His₅ tag. The sameresult was obtained for 12 other V_(H)s following a one-hour incubationwith trypsin. Mass spectrometry on a randomly selected sample of thetrypsin-treated V_(H)s (i.e., HVHP414, HVHP419, HVHP420, HVHP423,HVHP429, HVHP430 and HVHM81) confirmed that in every case the molecularmass of the digested product corresponded to a V_(H) with the c-Myc Lysas the C-terminal residue. HVHM41 gave a significantly shorter fragmentthan the rest upon digestion, and in this case mass spectrometryexperiments mapped the cleavage site to the Arg99 in CDR3 (data notshown).

Eleven V_(H)s ranging in concentration from 0.32 mg/ml (HVHP428) to 3.2mg/ml (HVHP420) were incubated at 37° C. for 17 days. Their stabilitywas subsequently determined in terms of oligomerization state andprotein A binding. As shown by gel filtration chromatography, treatmentof V_(H)s at 37° C. did not induce any aggregate formation: all V_(H)sgave chromatogram profiles which were virtually identical to those ofuntreated V_(H)s and stayed essentially as monomers (see the example forHVHP420; FIG. 4 c). To ensure that the V_(H)s maintained their nativefold following 37° C. treatment, two V_(H)s, namely, HVHP414 (1.2 mg/ml)and HVHP420 (3.2 mg/ml), were selected at random and their K_(D)s ofbinding to protein A were determined by SPR (Data shown for HVHP420;FIG. 4 c inset) and compared to the K_(D)s obtained for untreated V_(H)s(Table 2). The calculated K_(D)s for the heat-treated V_(H)s were 1.4 μMand 1.0 μM for HVHP414 and HVHP420, respectively. These values areessentially identical to the corresponding values for the untreatedV_(H)s (Table 2), demonstrating that 37° C. treatment of V_(H)s did notaffect their native fold. The possibility that V_(H)s may have been in aless compact, non-native fold during the 37° C.-incubation periods andresumed their native fold upon returning to room temperature during gelfiltration and SPR experiments is unlikely in light of the fact that theV_(H)s were resistant to trypsin at 37° C. (see above), a propertytypically associated for well folded native proteins.

The refolding efficiency (RE) of the human V_(H)s was investigated bycomparing the K_(D)s of the binding of the native (K_(D)n) andheat-treated, refolded (K_(D)ref) V_(H)s to protein A (Tanha, J. et al.,2002). When a fraction of the V_(H) is inactivated by heat treatment themeasured K_(D) would be higher, since this parameter is based on theconcentration of folded, i.e., active, antibody fragment. Thus, theratio of K_(D)n to K_(D)ref gives a measure of V_(H) RE. FIG. 5 comparessensorgrams for HVHP423 binding to immobilized protein A in native(thick lines) and refolded (thin lines) states at several selected V_(H)concentrations. As can be seen, binding of the refolded V_(H) to proteinA is less in all instances, indicating that the unfolding is not fullyreversible. For each of the 14 V_(H)s, protein A binding in both nativeand refolded states was measured at several concentrations, and theK_(D)s and subsequently REs were determined (Table 2; K_(D)ref valuesare not shown). The K_(D)s and REs of two anti-idiotypic llama V_(H)HS,H11F9 and H11B2, which were used as references, were also determined.Four V_(H)s had REs in the range of 92%-95%, similar to the REs forH11F9 and H11B2, 95% and 100%, respectively. Another five had REs in therange of 84%-88% and three over 70%. Only two had significantly lowerRE: HVHP413 (52%) and HVHP421 (14%). Several published V_(H)Hs examinedpreviously had RE around 50% (van der Linden, R. H. et al., 1999).

Human V_(H) phage display library construction and panning. cDNA wassynthesized from human spleen mRNA (Ambion Inc., Austin, TX) usingrandom hexanucleotide primers and First Strand cDNA^(TM) kit (GEHealthcare, Baie d′Urfé, QC, Canada). Using the cDNAs as template, V_(H)genes with flanking C_(H) sequences were amplified by polymerase chainreaction (PCR) in nine separate reactions using V_(H) framework region 1(FR-1)-specific primers and an immunoglobin M-specific primer (de Haard,J.J. et al., 1999). The products were gel-purified and used as thetemplate in the second round of PCT to construct V_(H) genes using theFR1- and FR4-specific primers (de Haard, H.J. et al., 1999) that alsointroduced flanking Apal I and Not I restriction sites for cloningpurposes. The resultant V_(H) repertoire DNAs were cloned intofd-tetGIIID phage vector and a V_(H) phage display library wasconstructed (Tanha, J., et al., 2001). Panning against protein A(Amersham Biosciences Inc.) was performed as described (Tanha, J. etal., 2001). Germline sequence assignment of the selected V_(H)s wasperformed using DNAPLOT software Version 2.0.1 and V BASE version 1.0Llama V_(H)Hs H11C7, H11F9 and H11B2 were isolated from a llama V_(H)Hphage display library by panning against H11 scFv as described (Tanha,J. Et al., 2002).

V_(H) expression and purification. V_(H)s were cloned into pSJF2expression vectors by standard cloning techniques (Sambrook, J. FritschE. F. and Maniatis T, 1989). Periplasmic expression of sdAbs andsubsequent purification by immobilized metal affinity chromatography(IMAC) were performed as described (Muruganandam, A. et al., 2002).Protein concentrations were determined by A₂80 measurements using molarabsorption coefficients calculated for each protein (Pace, C. N. et al.,1995). Gel filtration chromatography of the purified V_(H)s wasperformed on a Superdex 75 column (GE Healthcare) as described (Deng, S.J. et al., 1995).

Binding and refolding efficiency experiments. Equilibrium dissociationconstants (K_(D)s) and refolding efficiencies (REs) of V_(H)s/V_(H)Hswere derived from surface plasmon resonance (SPR) data collected withBIACORE 3000 biosensor system (Biacore Inc., Piscataway, N.J.). Tomeasure the binding of V_(H)s to protein A, 2000 resonance units (RUs)of protein A or a reference antigen-binding fragment (Fab) wereimmobilized on research grade CM5 sensor chips (Biacore Inc.).Immobilizations were carried out at concentrations of 25 μg/ml (proteinA) or 50 μg/ml (Fab) in 10 mM sodium acetate buffer pH 4.5, using theamine coupling kit provided by the manufacturer. To measure the bindingof the anti-idiotypic llama V_(H)Hs to H11 scFv, 4100 RUs of 50 μg/ml H1scFv or 3000 RUs of 10 μg/ml Se155-4 IgG reference were immobilized asdescribed above. In all instances, analyses were carried out at 25° C.in 10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA and 0.005% P20at a flow rate of 40 μl/min, and surfaces were regenerated by washingwith the running buffer. To determine the binding activities of therefolded proteins, V_(H)s or V_(H)Hs were denatured by incubation at 85°C. for 20 min at 10 μg/ml concentrations. The protein samples were thencooled down to room temperature for 30 min to refold and weresubsequently centrifuged in a microfuge at 14,000 rpm for 5 min at roomtemperature to remove any protein precipitates. The supernatants wererecovered and analyzed for binding activity by SPR as described above.For both folded and refolded proteins data were fit to a 1:1 interactionmodel simultaneously using BIAevaluation 4.1 software (Biacore Inc.) andK_(D)s were subsequently determined. REs were determined from

${RE} = {\frac{K_{D}n}{K_{D}{ref}} \times 100}$

Where K_(D)n is the K_(D) of the native protein and K_(D)ref is theK_(D) of the refolded protein.

Tryptic digest experiments. 3 μl of a freshly prepared 0.1 μg/μlsequencing grade trypsin (Hoffmann-La Roche Ltd., Mississauga, ON,Canada) in 1 mM HCl was added to 60 μg V_(H) in 100 mM Tris-HCl bufferpH 7.8. Digestion reactions were carried out in a total volume of 60 μlfor 1 h at 37° C. and stopped by adding 5 μl of 0.1 μg/μl trypsininhibitor (Sigma, Oakville, ON, Canada). Following completion ofdigestion, 5 μl was removed and analyzed by SDS-PAGE; the remaining wasdesalted using ZipTiP_(C4) (Millipore, Nepean, ON, Canada), eluted with1% acetic acid in 50:50 methanol:water and subjected to V_(H) massdetermination by MALDI mass spectrometry.

Protein stability studies at 37° C. Single-domain antibodies (sdAbs) at0.32-3.2 mg/ml concentrations were incubated at 37° C. in PBS buffer for17 days. Following incubation, the protein samples were spun down in amicrofuge at maximum speed for 5 min even in the absence of any visibleaggregate formation. The samples were then applied onto a Superdex 75size exclusion column (GE Healthcare) and the monomeric peaks werecollected for SPR analysis against protein A. SPR analyses wereperformed as described above except that 500 RUs of protein A orreference Fab was immobilized and that immobilizations were carried outat concentration of 50 μg/ml.

NMR experiments—V_(H) samples for NMR analysis were dissolved in 10 mMsodium phosphate, 150 mM NaCl, 0.5 mM EDTA, and 0.02% NaN₃ at pH 7.0.The protein concentrations were 40 μM-1.0 mM. All NMR experiments werecarried out at 298 K on a Bruker Avance-800 or a Bruker Avance-500 NMRspectrometer. One-dimensional (1D) ¹H NMR spectra were recorded with16,384 data points and the spectral widths were 8,992.81 Hz at 500 MHzand 17,605.63 Hz at 800 MHz, respectively. Two-dimensional ¹H-¹H NOESYspectra of 2,048×400 data points were acquired on a Bruker Avance-800NMR spectrometer with a spectral width of 11,990.04 Hz and a mixing timeof 120 ms. In all NMR experiments, water suppression was achieved usingthe WATERGATE method implemented through the 3-9-19 pulse train (Piotto,M. et al., 1992; Sklenar, V. et al., 1993). NMR data were processed andanalyzed using the Bruker XWINNMR software package. All PFG-NMRdiffusion measurements were carried out with the water-suppressed LEDsequence (Altieri, A. S. et al., 1995), on a Bruker Avance-500 NMRspectrometer equipped with a triple-resonance probe with three-axisgradients. One-dimensional proton spectra were processed and analyzedusing Bruker Xwinnmr software package. NMR signal intensities wereobtained by integrating NMR spectra in the methyl and methylene protonregion (2.3 ppm to −0.3 ppm) where all NMR signals were attenuateduniformly at all given PFG strengths.

Human V_(L) phage display library construction and panning. cDNAs weresynthesized from human spleen mRNA as described above for the humanV_(H)s. The cDNA was used as template in PCR to amplify V_(L) genes in50 μl reaction volumes using six V_(κ) back primers, 11 V_(λ) backprimers (de Haard, H. J. et al., 1999), four V_(κ) For primers and twoV_(λ) For primers (Sblattero, D. et al., 1998). The back and forwardprimers were modified to have flanking Apa LI and Not I restrictionsites, respectively, for subsequent cloning purposes. Forward primerswere pooled together in ratios which reflected their degree ofdegeneracy. V_(λ) genes were PCRed in 11 separate reactions using thepooled V_(λ) For primers and 11 individual V_(λ) back primers.Similarly, V_(λ) genes were amplified in 6 separate reactions using thepooled V_(κ) For primers and 6 individual V_(λ) back primers. The PCRproducts were pooled, gel purified and digested with Apa LI and Not Irestriction endonucleases. The library was constructed as described forhuman V_(H)s. Plaque PCR was performed on individual library coloniesand the amplified V_(L) genes were sequenced as described (Tanha, J. etal., 2003). Panning against protein L (Biolynx Inc., Brockville, ON,Canada) and germline sequence assignment of the selected V_(L)s wereperformed as described above for human V_(H) library.

V_(L) expression and purification. V_(L) expression, purification,concentration determination and gel filtration chromatography werecarried out as described for V_(H)s in “V_(H) expression andpurification.”.

Expression and purification of V_(L) and V_(H) pentamers. Specificprimers were used in a standard PCR to amplify HVHP328 V_(H) and HVLP335V_(L) genes. Standard cloning techniques were used to clone the HVHP328and HVLP335 genes in fusion with VT1B pentamerization domain gene in anexpression vector to yield HVHP328PVT2 and HVLP335PTV2 pentamers,(Zhang, J. et al., 2004). Pentamers were expressed and purified asdescribed (Zhang, J. et al., 2004). Protein concentrations weredetermined as above.

Surface plasmon resonance of V_(L)s. The binding kinetics for theinteraction of the V_(L)s to protein L were determined by SPR usingBIACORE 3000 biosensor system (Biacore, Inc., Piscataway, N.J.). 680 RUsof protein L or 870 RUs of a Fab reference were immobilized on researchgrade CM5 sensor chips (Biacore). Immobilizations were carried out at aprotein concentration of 50 μg/ml in 10 mM acetate buffer pH 4.5 usingthe amine coupling kit supplied by the manufacturer. All measurementswere carried out at 25° C. in 10 mM HEPES buffer pH 7.4, containing 150mM NaCl, 3 mM EDTA and 0.005% P20 at a flow rate of 50 μl/min or 100μl/min. Surfaces were regenerated by washing with the running buffer.Data were evaluated using the BIAevaluation 4.1 software (Biacore,Inc.).

Surface plasmon resonance of the pentameric V_(L) and V_(H). The bindingkinetics for the interaction of HVHP328PVT2 with protein A andHVLP335PTV2 with protein L were also determined by SPR. 520 RUs ofprotein A or a Fab reference were immobilized as above. For the V_(L)pentamer, the same surfaces prepared above were used. Measurements werecarried out as above but at a flow rate of 20 μl/min. Surfaces wereregenerated by washing with 50 mM HCl for 3 s. Data were evaluated asdescribed for the monomers.

Cell Microagglutination

A single S. aureus colony from a BHI plate was used to inoculate 15 mLof BHI media. The bacteria were grown overnight at 37° C. at 200 rpm. Inthe morning, the culture was spun down in a swinging bucket, SorvallRT6000B refrigerated centrifuge at 4000 rpm for 10 min, the supernatantwas removed and the cell pellet was re-suspended in PBS buffer. Thecells were re-spun, the supernatant was removed and the cell pellet wasre-suspended again in PBS buffer. The cells were diluted to an A₆₀₀ of1.0, and serial dilutions of the cells were spread on BHI plates at 37°C. for overnight growth. The cell titer was determined in the morning.An A₆₀₀ of 1.0 corresponded to 1.5×10⁹ cells ml⁻¹. Identical steps weretaken to prepare E. coli starin TG1 cells for subsequentmicroagglutination assays, except that the growth media was 2×YT. Theviable counts were similar, A₆₀₀1.0=2.1×10⁹ cells ml⁻¹.

To perform microagglutination assays, two fold dilutions of HVHP328PVT2in PBS were performed from wells 1 to 11 in a microtiter plate. Well 12(blank) had only PBS. The total volume in each well was 50 μl.Subsequently, 1×10⁸ S. aureus cells in 50 μl PBS was added to all wellsand the plate was incubated overnight at 4° C. To have a permanentrecord of the results, a picture was taken from the plate in themorning. For the pentamer control experiment, HVHP328PVT2 was replacedwith the V_(L) pentamer, HVLP335PTV2. In the cell control experiments,the same two sets of experiments were repeated with E. coli TG1 cells.

Identification and Sequence Analysis of Monomeric Human V_(L)s

Essentially the same selection method which was employed to isolatesoluble V_(H)s from a human V_(H) phage display library was applied to ahuman V_(L) library for isolating soluble, monomeric V_(L)s. A humanV_(L) library with a size of 3×10⁶ was constructed. Twenty four plaquesfrom the library titer plates were picked and their V_(L) genes werePCRed and sequenced. The sequences were diverse in terms of germ-lineorigin although 75% of the V_(L)s were of Vλ origin (data not shown).Three rounds of panning against protein L resulted in To performmicroagglutination assays, two fold dilutions of HVHP328PVT2 in PBS wereperformed from wells 1 to 11 in a microtiter plate. Well 12 (blank) hadonly PBS. The total volume in each well was 50 μl. Subsequently, 1×10⁸S. aureus cells in 50 μl PBS was added to all wells and the plate wasincubated overnight at 4° C. To have a permanent record of the results,a picture was taken from the plate in the morning. For the pentamercontrol experiment, HVHP328PVT2 was replaced with the V_(L) pentamer,HVLP335PTV2. In the cell control experiments, the same two sets ofexperiments were repeated with E. coli TG1 cells.

Identification and Sequence Analysis of Monomeric Human V_(L)s

Essentially the same selection method which was employed to isolatesoluble V_(H)s from a human V_(H) phage display library was applied to ahuman V_(L) library for isolating soluble, monomeric V_(L)s. A humanV_(L) library with a size of 3×10⁶ was constructed. Twenty four plaquesfrom the library titer plates were picked and their V_(L) genes werePCRed and sequenced. The sequences were diverse in terms of germ-lineorigin although 75% of the V_(L)s were of VA origin (data not shown).Three rounds of panning against protein L resulted in enrichment forlarge plaques. Thirty-nine of large plaques were sequenced and 32 uniquesequences were identified (FIG. 6). HVLP325, HVLP335 and HVLP351occurred at frequency of 3, 4 and 2, respectively. Except for HVLP389which is of lambda class (subgroup Vλ1, germline 1b), the remaining 31V_(L)s belonged to the Vκ class. Of the 31 kappa V_(L)s, 24 fall withinthe VκIII subgroup and 7 within the Vκ1 subgroup. Sixteen of the 24VκIII sequences utilize L6 germline sequence with the remainingutilizing A27, L2 and L6 germline sequences. The Vκ1 subgroup V_(L)s areoriginated from O2/O12 or A30 germline sequence. Noticeable mutationsoccurred at position 96. The germline amino acids at this position arearomatic and hydrophobic amino acids Trp, Phe, Tyr, Leu or Ile for kappaV_(L)s and Tyr, Val or Ala for lambda V_(L)s. But in the selected poolof kappa V_(L)s only 5 out of 31 have their germline amino acids atposition 96: HVLP325, HVLP349, HVLP388, HVLP3109 and HVLP393. 21 aminoacids at position 96 are charged of which 20 are positively-charged:Arg, Lys or His. Two amino acids are Pro, one Gln, one Ser and one Thr.Of seven kappa V_(L)s analyzed by gel filtration chromatography formonomericity, six which had Arg or Lys at position 96 were alsomonomers, whereas HVLP325 with the germline amino acid Leu at position96 formed aggregates (see below). Similarly, HVLP389 which was of thelambda class and had a germline mutation to Ser was also monomeric (seebelow). These data correlates the deviation from the germline aminoacids at position 96 (27 out of 32) with improved biophysical propertiesof V_(L)s such as monomericity.

Eighteen V_(L)s of the kappa class had their last three residues(105-107) replaced with amino acids Thr, Val and Leu which are onlyfound in lambda V_(L)s. These substitutions may have had a role inimproving the biophysical properties of the kappa V_(L)s, resulting inthe selection of the aforementioned V_(L)s over the parental clones withthe original kappa residues at position 105-107.

Characterization of the Human V_(L)s

Eight of the selected V_(L)s with different V germline origins wereexpressed in E. coli in one-liter cultures and purified: HVLP324,HVLP325, HVLP335, HVLP342, HVLP351, HVLP364, HVLP389 and HVLP3103 (Table6). All were expressed in good yields ranging from 6.2 mg for HVLP324 toaround 75 mg for HVLP335 and HVLP364.

The aggregation tendency of the human V_(L)s was assessed in terms oftheir oligomerization state by gel filtration chromatography. V_(L)swere subjected to Superdex 75 gel filtration chromatography at aconcentration of 0.6 mg/ml. All except HVLP325 were essentially free ofaggregates and gave symmetric single peaks with the mean apparentmolecular mass of 12.7 kDa (range, 6.2-19.2 kDa) (FIG. 7A and Table 3).This is in agreement with the expected molecular mass for monomericV_(L)s, 13.4-13.8 kDa. Variation in apparent molecular mass forsingle-domain antibodies has been reported previously (Jespers, L. etal., 2004a; (Stevens, F. J. et al., 1980). For HVLP325, the aggregatesformed 11% of the total protein (aggregate plus monomer). HVLP351,HVLP342, HVLP335 and HVLP3103, were still monomer when tested at theirhighest concentration available, i.e., 0.89 mg/ml, 1.0 mg/ml, 4.9 mg/mland 5.9 mg/ml, respectively (FIG. 7B)

V_(L)s were subjected to Superdex-75 chromatography prior to BIACOREanalysis and purified monomer peaks collected even in the absence of anyevidence of aggregated material. In SPR analysis, all selected V_(L)sbound to protein L (FIG. 8). This was not unexpected since the V_(L)swere isolated by panning against protein L. For all, the K_(D)s ofbinding to protein L were in 0.6-3 μM (Table 3). HVLP324 and HVLP342 hadadditional smaller K_(D)s, 10 nM and 40 nM, respectively. Low affinityand high affinity bindings of V_(L)s of VκI subgroup to protein L havebeen reported previously (Reference). Both, HVLP324 and HVLP342, belongto VκI subgroup (Table 3). As expected, the kinetic and equilibrium datawere consistent with the monomeric peak being indeed monomeric.

Binding Analyses of Pentamers

Bindings of HVHP328PVT2 pentamer to protein A and HVLP335PTV2 pentamerto protein L were determined by surface plasmon resonance (FIG. 9). Theassociation rates were independently calculated from plots of k_(obs)versus concentration. More than one dissociation rate (k_(d)) could becalculated due to the heterogeneity in multivalent binding amongst thepentamer population. Therefore, more than one equilibrium dissociationconstant, K_(D), could be obtained. HVHP328PTV2 and HVLP335PTV2 hadminimum K_(D)s of 2 nM and 200 μM, respectively (Table 4). With slowerk_(d)s, HVHP328PTV2 and HVLP335PTV2 had K_(D)s as low as 900 and 90 pM,respectively.

Pathogen Detection by V_(L)s and V_(H)s

The protein A and L binding activity of the V_(H)s and V_(L)s can beused to detect bacteria which have protein A and/or L on their surfaces.This is possible if the V_(H)s and V_(L)s are soluble and monomeric(lack of tendency to aggregate) such as the V_(H)s and V_(L)s here.Variable domains derived from antibodies which lack light chains such ascamelid heavy chain antibodies or nurse shark and wobbegong shark IgNARsare naturally soluble and monomeric. From these, those with protein Aand L binding activity can also be used to detect bacteria which haveprotein A and/or L on their surfaces. Protein A is present on thesurface of the pathogenic bacteria, Staphylococcus aureus. Thus, theV_(H)s with protein A binding activity such as the ones described herecan be used to detect S. aureus. We performed a microagglutination assayto detect the ability of HVHP328PVT2 V_(H) pentamer to bind to S.aureus. A constant number of bacterial cells were incubated withtwo-fold dilutions of HVHP328PVT2 in microtiter wells (wells 1-11) (FIG.10). Well 12 had buffer instead of the pentamer. If the V_(H)s bind tothe bacterial cells, then the pentamer because of its multimeric natureshould be able to cross-link the cells and results in cellagglutination. The agglutinated cells will appear as diffused cells in amicrotiter well (FIG. 10). In the absence of any binding, noagglutination should occur, hence no agglutination, and the cells willappear as a dot at the bottom of the well. As shown in FIG. 10, thepentamer binds to the S. aureus, since there is agglutination of cells.The agglutination is observed up to well 7. Beyond well 7 theconcentration of the pentamer is too low for binding, hence noagglutination. The control V_(L) pentamer does not show anyagglutination, demonstrating the specificity of the V_(H) pentamer to S.aureus (FIG. 10). The binding is also cell-specific since the V_(H)pentamer as expected does not agglutinate E. coli (TG1 strain) orSalmonella cells (data not shown). Similarly, the V_(L) monomers andV_(L) pentamers with protein L binding activity can be used for thedetection of bacteria, in particular pathogenic bacteria such asPeptostreptococcus magnus, which have protein L on their cells surface.

It is understood that the examples described above in no way serve tolimit the true scope of this invention, but rather are presented forillustrative purposes.

REFERENCE LIST

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TABLE 1 V_(H) sequence deviations from parental germline sequences V_(H)V/J germlines Amino acid deviation from V and FR4 germline sequencesHVHP44 DP47/JH4b L5V, Q105R HVHB82 DP47/JH6c E1Q, L5Q HVHP421 DP47/JH4bE1Q, V2L, L5Q, L11V, G16R HVHP419 DP47/JH4b E1Q, V2L, L5Q, T77S, R83G,K94R HVHP430 DP47/JH3b E1Q, L5V, V12I, Q13K, S31N, G52AS, L78V, A93V,K94R HVHP429 DP47/JH4 L5V, G10T, S30I, S31N, G42D, E46D, A50T, G52aN,S53N, S56A K75N, A84P, E85D HVHM41 DP47/JH3a E1Q, L5V, E6Q, G16R, T28A,S53G, G55D, S56H, M108L HVHM81 DP47JH3a L5V, E6Q, G16R, S30D, S31D,S35H, A50G, G55A, E85G, V89L, K94R HVHP428 V3-49/JH4b E1Q, V2L, V5Q,R16G, T23A, G30S, D31S, T60A, G73D, K83R, T84A, V89M, T93A HVHP420DP-38/JH4b E1Q, S35T, S52aT HVHP414 DP-38/JH3b E1D, E6Q, A23T, T28P,K52T, A60V HVHP423 V3-53/JH1 E1Q, V2M, E6Q, L11V, I12V, N32S, Y33R,V37F, K43M, K64R, T68S, V89L HVHP44B V3-53/JH1 E1Q, E6Q, N32S, Y33R,V37F, K43M, Y58S, K64R, T68S, V89L HVHP413 YAC-5/JH3b E1Q, E6Q, Q13K,V29F, S31D, N32Y, V50F HVHP426 8-1B/JH3b E1Q, E6Q, L11V, G16R, T28I,S30D, S31G, N32Y, Y33A, S35H, K43Q, I51T, Y52N, S53N, Y58S, L78V

TABLE 2 Biophysical characteristics of the human V_(H)s TrypsinV_(H)/V_(H)H Exp. # (mg) K_(D) (μM) resistance RE (%) HVHP44 8.2 1.3 ✓93 HVHB82 5.9 0.2 ✓ 71 HVHP421 5.5 1.0 ✓ 14 HVHP419 3.4 1.6 ✓ 84 HVHP4306.4, 23.7 2.3 ✓ 88 HVHP429 3.4 1.3 ✓ 86 HVHM41 1.8 0.5 X 92 HVHM81 4.31.3 ✓ 87 HVHP428 3.1 1.8 ✓ 95 HVHP420 59.0 1.2 ✓ 92 HVHP414 11.8 1.6 ✓73 HVHP423 2.4, 62.1 3.0 ✓ 86 HVHP413 5.8 0.3 ✓ 52 HVHP426 6.3 0.8 ✓ 70H11F9* ND 3.5 ND 95 H11B2* ND 2.0 ND 100 # expression yield per liter ofbacterial culture *K_(D) s and RE s were determined against H11 scFv.

TABLE 3 Characteristics of the human V_(L)s Expression^(a) K_(D)Oligomerization V_(L) Subgroup mg μM state^(b) HVLP324 VκI 6.9 0.2,0.01^(c) Monomer HVLP325 VκIII 6.2 1 Monomer/Aggregate HVLP335 VκIII73.5 2 Monomer HVLP342 VκI 7.7 0.6, 0.04^(c) Monomer HVLP351 VκIII 8.9 2Monomer HVLP364 VκIII 77.1 3 Monomer HVLP389 VλI 16.7 1 Monomer HVLP3103VκIII 19.0 1 Monomer ^(a)Expression yield per liter of bacterialculture. ^(b)Oligomerization state was determined by gel filtrationchromatography. ^(c)The smaller K_(D) values correspond to the bindingof the of HVLP324 and HVLP342 to the high affinity sites on protein L.

TABLE 4 Kinetic and equilibrium constants for the bindings ofHVHP328PTV2 and HVLP335PTV2 to protein A and L, respectively PentabodyHVHP328PTV2 HVLP335PTV2 k_(a) (M⁻¹s⁻¹) 4.3 × 10⁵ 1.7 × 10⁶ k_(d) (s⁻¹) <1 × 10⁻³  <4 × 10⁻⁴ K_(D) (M)  <2 × 10⁻⁹  <2 × 10⁻¹⁰

1. A method of identifying stable human or humanized target VH and/or VLantibody fragments, comprising: (a) obtaining a VH and/or VL phagedisplay library and reducing the library size, yielding a phage displaylibrary expressing a variety of VH and/or VL antibody fragments, whereinthe library size is reduced by panning against a target antigen, panningagainst a non-specific binder, or chromatography using a hydrophobicinteraction column or otherwise removing wild type phage without insertsfrom the library; (b) allowing infection of a bacterial lawn by thelibrary phage; (c) identifying phage which form larger than averageplaques on the bacterial lawn; (d) isolating the larger plaque phage instep (c); and (e) determining the sequence or other characteristics ofthe VH and/or VL antibody fragments expressed by the phage forminglarger plaques, wherein the human or humanized VH or VL antibodyfragment expressed in the larger plaque phage isolated in step (d) i) issoluble, ii) is monomeric, iii) is nonaggregating, iv) has relativelyhigh thermal refolding efficiency v) has relatively high meltingtemperature, vi) maintains its functionality following long incubationat 37° C., vii) is relatively resistant to chemical denaturants, viii)is relatively resistant to proteases, ix) has a relatively long shelflife at or above room temperature, at 4° C., or below 0° C., or x) anycombination of the above; when compared to a human or humanized VH or VLantibody fragment expressed in average plaque-forming phage.
 2. A methodas claimed in claim 1, where the phage is a filamentous phage.
 3. Amethod as claimed in claim 2, where the phage is M13 or fd.
 4. A methodas claimed in claim 1, wherein the human or humanized VH and/or VLantibody fragments are human VH or VL antibody fragments.
 5. An isolatedstable VH or VL antibody fragment identified by the method of claim 1.6. A VH or VL antibody fragment as claimed in claim 5 that is of humanorigin.
 7. A VH antibody fragment as claimed in claim 5 having an aminoacid sequence that deviates from the corresponding parental germlinesequence as shown in Table
 1. 8. A VH antibody fragment as claimed inclaim 5 belonging to the VH3 family.
 9. A VH antibody fragment asclaimed in claim 5 belonging to a V germline selected from the groupconsisting of DP47, V3-49, DP-38, V3-53, YAC-5 and 8-1 B.
 10. A VHantibody fragment as claimed in claim 5 belonging to a J germlineselected from the group consisting of JH4b, JH6c, JH3b, JH4, JH3a, andJH1.
 11. A VH antibody fragment as claimed in claim 5 and having aresidue other than valine at position 37 of its amino acid sequence. 12.A VH antibody fragment as claimed in claim 9 having a phenylalanine ortyrosine residue at position 37 of its amino acid sequence.
 13. A VHantibody fragment as claimed in claim 5 having a mutation at a positionselected from the group consisting of positions 1, 5, 6, 35, 83, 84, 84aand 108 of its amino acid sequence.
 14. A VH antibody fragment asclaimed in claim 5 that binds to protein A.
 15. A VL antibody fragmentas claimed in claim 5 belonging to subgroup Vκ1, Vκ3 or Vλ1.
 16. A VLantibody fragment as claimed in claim 5 belonging to the kappa class.17. A VL antibody fragment as claimed in claim 5 belonging to a Vgermline selected from the group consisting of L6, A27, L2, L16, O2/O12,A30 and 1b.
 18. A V L antibody fragment as claimed in claim 5 belongingto a J germline selected from the group consisting of Jκ1, Jκ4, Jκ2, andJ3λb.
 19. A VL antibody fragment as claimed in claim 5 having anarginine, proline, lysine, threonine, leucine, serine, tyrosine,glutamic acid, glutamine or histidine residue at position 96 of itsamino acid sequence.
 20. A VL antibody fragment as claimed in claim 5having a charged amino acid residue at position 96 of its amino acidsequence.
 21. A VL antibody fragment as claimed in claim 5 that binds toprotein L.
 22. A VL antibody fragment as claimed in claim 5 having athreonine, valine or leucine amino acid residue at positions 105, 106and 107 of its amino acid sequence.
 23. A pharmaceutical compositioncomprising the antibody fragment of claim 5 and a pharmaceuticallysuitable agent.